Rapid-acting insulin compositions

ABSTRACT

The invention is a pharmaceutical composition of human insulin or insulin analog that includes citrate, treprostinil and stabilizing agents, that has faster pharmacokinetic and/or pharmacodynamic action than commercial formulations of existing insulin analog products and that is stable for commercial use.

This application is a continuation of U.S. application Ser. No.15/241,412 (now U.S. Pat. No. 9,901,623), filed Aug. 19, 2019, whichclaims priority to U.S. Provisional Application No. 62/210,469 filedAug. 27, 2015.

The present invention is a pharmaceutical composition for use in thetreatment of diabetes to counteract postprandial blood glucoseexcursions and for acute treatment of hyperglycemia. The composition,which includes human insulin or an insulin analog, citrate, treprostiniland stabilizing agents, has a faster uptake of insulin from injectionsites than existing commercial insulin compositions and that is stablefor commercial use. The composition is useful for providing meal-timeinsulin activity, for use in continuous subcutaneous insulin infusion(CSII) or as an acute treatment for hyperglycemia when insulin isneeded.

The time-action profile of insulin is important for controllingpostprandial blood glucose levels. In healthy individuals, the pancreassecretes a spike of insulin in response to absorbed food, which resultsin increased plasma insulin levels within several minutes. Inindividuals with type 1 diabetes and in certain individuals with type 2diabetes, insulin must be administered; however, administered insulinenters blood slowly from the subcutaneous space. Delayed release ofinsulin and onset of action which is too slow at the beginning of a mealleads to hyperglycemia during or immediately after the meal. Aprotracted duration of action from the subcutaneous space leads toexcessive insulin between meals which can cause postprandialhypoglycemia.

There have been previous efforts to accelerate the time action ofinsulin products. Early efforts to develop such products included thedevelopment of novel rapid-acting insulin analogs, like insulin lispro(HUMALOG®), insulin aspart (NOVOLOG®), and insulin glulisine (APIDRA®).Insulin lispro and insulin aspart achieve rapid action through changesin the amino acid sequences from human insulin that weaken thedimer-dimer interface and alter hexameric stability under subcutaneousconditions. Insulin glulisine also includes changes in the sequences ofthe amino acid chains in human insulin; however, its commercialformulation lacks zinc and does not form stabilizing hexamers. Anotherinsulin analog stated to be rapid-acting, but which is still indevelopment, Fluorolog, includes a single fluorine atom attached to thePhe residue at position 24 of the insulin B-chain.

The rapid-acting insulin analogs insulin lispro, aspart and glulisinebecame available in the 1990s and early 2000s. Even with so-calledrapid-acting insulin analogs, however, the maximum plasma insulin levelis not reached until 50-90 minutes following a subcutaneous injection.This is slower than endogenous insulin release from a normallyfunctioning pancreas and does not always match glucose absorptionprofiles.

Another avenue to achieve rapid action that has been explored is the useof ingredients or excipients which improve the time action profile ofinsulin when provided in combination with insulin. For example, U.S.Pat. No. 8,324,157 states that a faster onset of action compared withexisting insulin therapies can be achieved by adding a nicotiniccompound, such as nicotinamide, and the amino acid arginine, andoptionally glutamic acid. US2013/0231281 discloses compositionscomprising an insulin and oligosaccharides, either alone or incombination with a polyanionic compound and states that suchcompositions are fast acting. US2014/0113856 discloses compositionscontaining insulin in combination with a zinc chelator, such asethylenediaminetetraacetic acid (EDTA), another excipient described inthe publication as a “dissolution/stabilization” agent, such as citricacid or sodium citrate, and a magnesium-containing compound, and statesthat such compositions have more rapid absorption rates and declinesfrom peak concentrations. US2015/0065423 describes compositionscomprising a peptide and a vasodilatory agent, discloses lists ofvasodilatory agents from three different categories, and provides dataon compositions containing insulin lispro and nitroglycerin and statesthat such formulations are rapid acting.

Despite these and other efforts, a need remains for insulin compositionsthat have more rapid uptake of insulin into the blood from the injectionsite, more rapid onset and/or offset of action than existing insulinproducts, and chemical and physical stability during storage and useconditions. The present invention seeks to provide compositions whichmeet one or more of these needs.

It has surprisingly been found that compositions containing certainconcentrations of both citrate and treprostinil have a more rapid timeaction profile than existing commercially available insulincompositions, and that the chemical and physical stability of thecompositions containing those concentrations of both citrate andtreprostinil can be maintained, without eliminating the improvements intime action, by including in the compositions zinc and one or moreadditional stabilizing agents such as a surfactant, magnesium chlorideor sodium chloride.

Accordingly, the present invention provides pharmaceutical compositionscomprising: an insulin; citrate, in a concentration from about 5 toabout 25 mM; treprostinil, in a concentration from about 0.04 to about20 μg/mL; zinc, in a concentration sufficient to provide at least 2 zincions per six molecules of insulin; a preservative; and one or moreadditional stabilizing agents; and having a pH of about 7.0 to about 7.8at room temperature.

In certain embodiments, the pharmaceutical composition comprises insulinlispro, in a concentration from about 100 to about 200 U/mL; citrate, ina concentration from about 15 to about 25 mM; treprostinil, in aconcentration from about 0.5 to about 2 μg/mL; zinc, in a concentrationfrom about 0.2 to about 1 mM; m-cresol, in a concentration from about2.5 to about 3.8 mg/mL; poloxamer 188, in a concentration from about0.03 to about 0.12 w/v; magnesium chloride, in a concentration resultingin a molar ratio of magnesium chloride to citrate of about 1:3;glycerol, in a concentration from about 1 to about 2 mg/mL; and has a pHfrom about 7.0 to about 7.8 at room temperature.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 100 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.6 mM; poloxamer 188, in aconcentration of about 0.09% w/v; magnesium chloride, in a concentrationof about 5 mM; m-cresol, in a concentration of about 3.15 mg/mL;glycerol, in a concentration of about 1.61 mg/mL; and has a pH of about7.4.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.8 mM; poloxamer 188, in aconcentration of about 0.06 to 0.12% w/v; magnesium chloride, in aconcentration of about 5 mM; m-cresol, in a concentration of about 3.15mg/mL; glycerol, in a concentration of about 1.61 mg/mL; and has a pH ofabout 7.4.

In certain embodiments, the pharmaceutical composition comprises insulinlispro, in a concentration from about 100 to about 200 U/mL; citrate, ina concentration from about 15 to about 25 mM; treprostinil, in aconcentration from about 0.5 to about 2 μg/mL; and zinc, in aconcentration from about 0.2 to about 2 mM.

In certain embodiments, the pharmaceutical composition comprises insulinlispro, in a concentration from about 100 to about 200 U/mL; citrate, ina concentration from about 15 to about 25 mM; treprostinil, in aconcentration from about 0.5 to about 2 μg/mL; zinc, in a concentrationfrom about 0.2 to about 2 mM; and magnesium, in a concentrationresulting in a molar ratio of magnesium to citrate of about 1:1 to about1:5.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 100 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.6 mM; and magnesium, in aconcentration of about 5 mM.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 100 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.6 mM; magnesium, in aconcentration of about 5 mM; and m-cresol, in a concentration of about3.15 mg/mL.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 100 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.6 mM; magnesium, in aconcentration of about 5 mM; m-cresol, in a concentration of about 3.15mg/mL; and glycerol, in a concentration of about 12 mg/mL.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 100 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.6 mM; magnesium, in aconcentration of about 5 mM; m-cresol, in a concentration of about 3.15mg/mL; and a total chloride concentration of about 10 to about 50 mM;and has a pH of about 7.4

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 100 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.6 mM; magnesium, in aconcentration of about 5 mM; m-cresol, in a concentration of about 3.15mg/mL; glycerol, in a concentration of about 12 mg/mL; and a totalchloride concentration of about 10 to about 50 mM.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 100 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.6 mM; magnesium, in aconcentration of about 5 mM; m-cresol, in a concentration of about 3.15mg/mL; glycerol, in a concentration of about 12 mg/mL; and a totalchloride concentration of about 10 to about 50 mM; and has a pH of about7.4.

In certain embodiments, the pharmaceutical composition comprises insulinlispro, in a concentration of about 100 U/mL; citrate, in aconcentration of 15 mM; treprostinil, in a concentration of 1 μg/mL;zinc, in a concentration of about 0.3 mM; phosphate, in a concentrationof about 7 mM; glycerol, in a concentration of about 16 mg/mL at pH 7.4.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; and zinc, in a concentration of about 0.7 to about 1.7 mM.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.8 mM; and magnesium in aconcentration of about 5 to about 10 mM.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.8 mM; and magnesium in aconcentration of about 5 mM.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.8 mM; magnesium in aconcentration of about 5 to about 10 mM; and a total chlorideconcentration of about 10 to about 50 mM.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.8 mM; magnesium in aconcentration of about 5 to about 10 mM; and glycerol in a concentrationof about 12 mg/mL.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.8 mM; magnesium in aconcentration of about 5 to about 10 mM; glycerol in a concentration ofabout 12 mg/mL; and a total chloride concentration of about 10 to about50 mM.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.7 to about 1.7 mM; andm-cresol, in a concentration of about 3.15 mg/mL.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.8 mM; magnesium in aconcentration of about 5 to about 10 mM; and m-cresol in a concentrationof about 3.15 mg/mL.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.8 mM; magnesium in aconcentration of about 5 to about 10 mM; a total chloride concentrationof about 10 to about 50 mM; and m-cresol, in a concentration of about3.15 mg/mL.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.8 mM; magnesium in aconcentration of about 5 to about 10 mM; glycerol in a concentration ofabout 12 mg/mL; and m-cresol, in a concentration of about 3.15 mg/mL.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.8 mM; magnesium in aconcentration of about 5 to about 10 mM; glycerol in a concentration ofabout 12 mg/mL; and a total chloride concentration of about 10 to about50 mM; and m-cresol, in a concentration of about 3.15 mg/mL.

In certain embodiments, the pharmaceutical composition comprises:insulin lispro, in a concentration of about 200 U/mL; citrate, in aconcentration of about 15 mM; treprostinil, in a concentration of about1 μg/mL; zinc, in a concentration of about 0.8 mM; magnesium in aconcentration of about 5 mM; glycerol in a concentration of about 12mg/mL; and a total chloride concentration of about 10 to about 50 mM;and m-cresol, in a concentration of about 3.15 mg/mL.

In addition, the present invention also provides a method of treatingdiabetes comprising administering to a human in need thereof aneffective dose of a pharmaceutical composition of the present invention.

In addition, the present invention provides a pharmaceutical compositionfor use in therapy. More particularly, the present invention provides apharmaceutical composition for use in the treatment of diabetes. Thepresent invention also provides the use of a pharmaceutical compositionin the manufacture of a medicament for the treatment of diabetes.

In addition, the present invention provides an article of manufacturecomprising a pharmaceutical composition. More particularly, in certainaspects the article of manufacture is a multi-use vial, a cartridge, are-usable pen injector, a disposable pen device, a pump device forcontinuous subcutaneous insulin infusion therapy or a container closuresystem for use in a pump device for continuous subcutaneous insulininfusion therapy.

When used herein, the term “composition” refers to a combination ofinsulin and the other ingredients or excipients wherein the insulin andother ingredients or excipients are in a single combined formulation,typically an aqueous formulation.

When used herein, “insulin” means human insulin or a rapid-actingstructural variant, mutein, or analog of human insulin that has thefunctional activity of but faster onset of action than human insulin.Particular rapid-acting analogs of human insulin are insulin lispro,insulin aspart, and insulin glulisine. Insulin for commercial productsmay be produced using recombinant DNA methods or by chemical synthesis.Recombinant methods are well-known and are strongly preferred. Amolecule of human insulin (CAS No. 11061-68-0) consists of two aminoacid chains, A and B, whose sequences are well-known.

The human insulin A-chain has the following sequence of amino acids:

(SEQ ID NO: 1) Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn.

The human insulin B-chain has the following sequence of amino acids:

(SEQ ID NO: 2) Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr.The chains are joined by two disulfide bonds: CysA7-CysB7 andCysA20-CysB19.The A-chain has an intra-chain disulfide bond at CysA6-CysA11. Humaninsulin has the empirical formula C₂₅₇H₃₈₃N₆₅O₇₇S₆ and a molecularweight of 5808.

Insulin lispro, the drug substance in HUMALOG®, is identical to humaninsulin in terms of its primary amino acid sequence except for aninversion of the natural proline-lysine sequence on the B-chain atpositions 28 and 29 (28^(B)-L-Lysine-29^(B)-L-proline human insulin).Insulin lispro (CAS No. 133107-64-9) has been shown to be equipotent tohuman insulin on a molar basis but its effect after subcutaneousinjection is more rapid and of shorter duration than that of injectedsoluble human insulin. HUMALOG® contains m-cresol as a preservative anda stabilizing agent, a tonicity modifier (glycerol), a buffering agent(dibasic sodium phosphate), a stabilizer (zinc oxide) and pH adjustmentfor the vehicle.

A molecule of insulin lispro consists of the human insulin A-chain (SEQID NO. 1) cross-linked with the insulin lispro B-chain, whose amino acidsequence is given by SEQ ID NO:3, below:

(SEQ ID NO: 3) Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Lys Pro Thr.The chemical formula of insulin lispro is C₂₅₇H₃₈₃N₆₅O₇₇S₆ and itsmolecular weight is approximately 5808. One unit of insulin lispro isequivalent to 0.0347 mg insulin lispro.

Insulin aspart (CAS No. 116094-23-6), the drug substance in NOVOLOG®, isanother rapid-onset insulin analog. Its structure consists of theA-chain of human insulin (SEQ ID NO: 1) and a B-chain in which the Proat B28 is replaced with Asp (Pro-B28-Asp human insulin), as reflected inthe following amino acid sequence:

(SEQ ID NO: 4) Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Lys Thr.Insulin aspart (28^(B) aspartic acid-human insulin) has the empiricalformula C₂₅₆H₃₈₁N₆₅O₇₉S₆ and a molecular weight of about 5826. One unitof insulin aspart corresponds to 6 nmol, corresponding with 0.035 mgsalt-free anhydrous insulin aspart.

Insulin glulisine (CAS No. 207748-29-6), the drug substance in APIDRA®,is yet another rapid-onset insulin analog. A molecule of insulinglulisine consists of human insulin A-chain (SEQ ID NO: 1) and amodified B-chain (Asn-B3-Lys, Lys-B29-Glu) compared with human insulin,as reflected in the following amino acid sequence:

(SEQ ID NO: 5) Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Glu Thr.

Insulin glulisine (3^(B)-lysine-29^(B)-glutamic acid-human insulin) hasthe empirical formula C₂₅₈H₃₈₄N₆₄O₇₈S₆ and a molecular weight of 5823.One unit of insulin glulisine corresponds approximately to 0.0349 mg ofinsulin glulisine.

The following scheme depicts the amino acid sequences and disulfidebonds of human insulin and of the rapid-acting insulin analogs that arepresently approved for use in treating meal-time excursions of bloodglucose:

In certain embodiments, the compositions of the present invention haveconcentrations of insulin from about 40 to about 500 U/mL. In certainembodiments, the compositions of the present invention haveconcentrations of insulin from about 100 to about 500 U/mL. In certainembodiments, the compositions of the present invention haveconcentrations of insulin from about 100 to about 300 U/mL. In certainembodiments, the compositions of the present invention haveconcentrations of insulin from about 100 to about 200 U/mL. In certainpreferred embodiments, the compositions comprise about 100 U/mL or about200 U/mL.

In an embodiment, the insulin is selected from the group consisting ofhuman insulin, or a rapid-acting structural variant, mutein, or analogof human insulin, such as insulin lispro, insulin aspart or insulinglulisine. In a preferred embodiment, the insulin is insulin lispro.

The improvements in the time action profile of the insulin demonstratedin the present invention are achieved through the use of certainspecific concentrations of citrate and treprostinil.

The term “citrate” refers to any compound containing the citrate ion,which has the chemical name 2-hydroxypropane-1,2,3-tricarboxylate,molecular formula C₆H₅O₇ ⁻³, and molecular weight of 189. The citrateion is widely distributed in plants and animals and is a naturallyoccurring component of the diet. It is a common metabolite in oxidativemetabolism and an important component of bone. A number of citrates areGRAS (generally regarded as safe) by the U.S. Food and DrugAdministration for use in foods, including the following:

GRAS Substance Formula (m.w.) CAS No. 21 CFR Citric acid C₆H₈O₇ (192.12)77-92-9 184.1033 Sodium citrate C₆H₅Na₃O₇ (258.07) 68-04-2 184.1751Potassium citrate C₆H₅O₇K₃ (324.41) 6100-05-6 184.1625 monohydrate

Various citrate-containing compounds are also included in parenteraldrug products according to the U.S. Food and Drug AdministrationInactive Ingredients database, including for example, citric acid,citric acid monohydrate, citric acid anhydrous, sodium citrate,anhydrous trisodium citrate, trisodium citrate dihydrate. The particularcitrate compound used in the compositions of the present invention maybe the acidic form or various salt forms, especially the alkali (e.g.,sodium and potassium) salts and/or mono or dihydrates thereof. Of these,sodium citrate is preferred.

It has been found that the concentration of citrate which may be used incompositions that are both fast acting and stable ranges from about 5 toabout 25 mM. Certain compositions have citrate concentrations of about15, about 20 or about 25 mM. It has been found that higherconcentrations of citrate may lead to greater improvements in timeaction, but also may lead to greater liabilities from a stabilitystandpoint. Thus, compositions with citrate concentrations at the upperend of the range require additional stabilizing agents in order to havechemical and physical stability for long-term storage and use, asdescribed in more detail below.

Treprostinil is a synthetic analog of prostacyclin, and has the chemicalname(1R,2R,3aS,9aS)-[[2,3,3a,4,9,9a-Hexahydro-2-hydroxy-1-[(3S)-3-hydroxyoctyl]-1H-benz[f]inden-5-yl]oxy]aceticacid (CAS No. 81846-19-7), molecular weight of 390.52 and a molecularformula of C₂₃H₃₄O₅. Treprostinil is the active ingredient in thecommercial drug products sold under the trade names Remodulin®, Tyvaso®and Orenitran™, which are indicated for the treatment of pulmonaryarterial hypertension to diminish symptoms associated with exercise(Remodulin®) and to improve exercise ability (Tyvaso® and Orenitran™).Tyvaso® and Orenitran™ are, respectively, inhalation and oral dosageforms, and Remodulin® is indicated for subcutaneous or intravenous useas a continuous infusion. Remodulin® is currently available in 1, 2.5, 5and 10 mg/mL dosage strengths, and each mL contains 3 mg m-cresol, 6.3mg sodium citrate, either 5.3 mg (1, 2.5 and 5 mg/mL strengths) or 4.0mg (10 mg/mL strength strength) sodium chloride, and water forinjection.

Like citrate, treprostinil is included to contribute to the improvementin the time action profile of the insulin. Unlike citrate, however, anincrease in the concentration of treprostinil has not been found to havea negative impact on stability. Due to treprostinil's potentvasodilatory effects, however, the concentration of treprostinil incompositions of the present invention must not be so high as to causeundesired systemic effects.

Moreover, the amount of insulin, and thus the volume of the composition,administered to a given subject at a given time is titrated based uponthe subject's blood glucose levels and/or anticipated carbohydrateintake. As a result, the total quantity of treprostinil provided willvary from injection to injection. For example, in some circumstances adiabetic may wish to have as little as 1 unit of insulin administered,which would be a total injection volume of just 10 μL from a 100 U/mLinsulin lispro composition. On the other hand, currently availableinjection devices provide for doses as high as 80 U in a singleinjection—the total volume of such a dose from a 100 U/mL compositionwould be over an order of magnitude higher than that of the 1 unit dose,and some type 2 diabetes patients may require a dose of more than 100insulin units, usually requiring more than one injection.

Thus, in compositions wherein insulin and treprostinil are both presentin a single combined formulation, the treprostinil concentration must besufficient to contribute to improvements in time action, even when arelatively small dose of insulin is needed, but must not be so high asto cause undesired systemic effects when a relatively high dose ofinsulin is needed. In certain embodiments, the composition comprisestreprostinil in a concentration from about 0.1 to about 50 μM, or about0.04 to about 20 μg/mL. In certain embodiments, the compositioncomprises treprostinil in a concentration from about 0.04 to about 10μg/mL. A preferred treprostinil concentration in insulin compositionshaving insulin concentrations ranging from about 100 to about 200 U/mLis from about 0.5 to about 2 μg/mL. In certain embodiments, thetreprostinil concentration is about 1 μg/mL.

As described above, while the addition of citrate leads to improvementsin time action, it may also contribute to greater liabilities from astability standpoint. Thus, the compositions of the present inventionrequire one or more stabilizing agents above and beyond those includedin currently available commercial formulations of rapid acting insulinanalogs, such as excess zinc, surfactants, magnesium-containingcompounds, such as magnesium chloride, and chloride-containingcompounds, such as magnesium chloride and/or sodium chloride.

With regards to zinc, the compositions of the present invention must, ata minimum, include zinc in a concentration provides at least enough zincions for the insulin molecules to form stabilizing hexamers, which have2 specific, high affinity zinc binding sites. See, e.g., BioMetals18:295-303 (2005), available athttp://rd.springer.com/article/10.1007/s10534-005-3685-y. The zinc ionsincorporated into such insulin hexamers are sometimes referred to as“bound” zinc. Thus, the compositions of the present invention mustinclude sufficient zinc to provide at least 2 ions of zinc per hexamerof insulin. In certain embodiments of the present invention having, forexample, insulin concentrations of about 100 U/mL, about 200 U/mL, about300 U/mL or about 500 U/mL, the minimum zinc concentration necessary toprovide 2 ions of zinc per insulin hexamer would be about 0.2 mM, about0.4 mM, about 0.6 mM or about 1 mM, respectively.

The inclusion of excess zinc—i.e., more zinc than would be bound in the2 specific, high affinity zinc binding sites in insulin hexamersdescribed above—however may be used to further stabilize thecomposition. Such zinc is sometimes referred to as “free” or “unbound”zinc. Currently available zinc-containing formulations include betweenabout 2 and 4 zinc ions per hexamer of insulin. For example, the 100U/mL formulations of insulin lispro (HUMALOG®) and insulin aspart(NOVOLOG®) have about 3 ions of zinc per six molecules of insulin, whichcorresponds with a concentration of about 0.3 mM. The currentlyavailable 200 U/mL formulation of HUMALOG® has about 3.5 ions of zincper six molecules of insulin, which corresponds with a zincconcentration of about 0.7 mM. The currently available 100 U/mLformulation of human insulin sold by Eli Lilly and Company (HUMULIN® R)contains about 2.3 ions of zinc per six molecules of insulin, whichcorresponds with a zinc concentration of about 0.23 mM.

In certain compositions of the present invention, the inclusion ofexcess free or unbound zinc—i.e., zinc which is not bound in the 2specific, high affinity zinc binding sites in insulin hexamers describedabove—has been found to have a stabilizing effect. Compositions havingabout 100 U/mL of insulin lispro and zinc concentrations up to about 1mM—which would constitute about 0.2 mM bound and about 0.8 mM unbound orfree zinc—have been found to be both fast acting and stable. Theinclusion of too much free or unbound zinc, however, may attenuate theimprovements in time action. For example, a composition having about 100U/mL of insulin lispro with a zinc concentration of about 5 mM—whichwould constitute about 4.8 mM unbound zinc—was found to not have theimprovements in time action seen in compositions with lower zincconcentrations. In certain embodiments, the concentration of zinc rangesfrom about 0.2 to about 2 mM, about 0.3 to about 1.7 mM, about 0.7 toabout 1.7 mM, about 0.4 to about 1 mM, about 0.4 to about 0.8 mM orabout 0.6 to about 0.9 mM. In certain embodiments the composition ofzinc is about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about0.8, about 0.9, about 1, about 1.25 or about 1.7 mM. In certainembodiments comprising about 100 U/mL insulin lispro, the concentrationof zinc is about 0.6 mM. In certain embodiments comprising about 200U/mL insulin lispro, the concentration of zinc is about 0.8 mM.

Another stabilizing agent which may be used is a surfactant. Examples ofsurfactants disclosed for use in parenteral pharmaceutical compositionsinclude polysorbates, such as polysorbate 20 (TWEEN® 20), polyethyleneglycols such as PEG 400, PEG 3000, TRITON™ X-100, polyethylene glycolssuch as polyoxyethylene (23) lauryl ether (CAS Number: 9002-92-0, soldunder trade name BRIJ®), alkoxylated fatty acids, such as MYRJ™,polypropylene glycols, block copolymers such as poloxamer 188 (CASNumber 9003-11-6, sold under trade name PLURONIC® F-68) and poloxamer407 (PLURONIC® F127), sorbitan alkyl esters (e.g., SPAN®),polyethoxylated castor oil (e.g., KOLLIPHOR®, CREMOPHOR®) and trehaloseand derivatives thereof, such as trehalose laurate ester. In certainembodiments, the surfactant is selected from the group consisting ofpolyoxyethylene (23) lauryl ether, poloxamer 188 and trehalose laurateester. Most preferred is poloxamer 188. In certain embodiments, theconcentration of surfactant ranges from about 0.003 to about 2% w/v,about 0.003 to about 0.3% w/v or about 0.01 to about 0.2% w/v. Inpreferred embodiments wherein the surfactant is poloxamer 188, theconcentration of poloxamer 188 ranges from about 0.06 to about 0.12 w/v.In certain embodiments, the concentration of poloxamer 188 is about0.06% w/v. In other embodiments, the concentration of poloxamer 188 isabout 0.09% w/v. In other embodiments, the concentration of poloxamer188 is about 0.12% w/v.

Another stabilizing agent which may be used in compositions of thepresent invention is magnesium, which may be provided for examplethrough the addition of a magnesium-containing compound such asmagnesium chloride, which has a molecular formula of MgCl₂ and molecularweight of 95.211. While MgCl₂ may have stabilizing effects in certaincompositions, high chloride ion (Cl⁻) concentrations may result ininsulin crystallization at low temperatures, and magnesium (Mg⁺²)concentrations which exceed the concentration of citrate will result ininsulin precipitation. Thus, the maximum amount of magnesium chloridethat may be included is limited by the amount of citrate that isincluded. In certain embodiments, when MgCl₂ is used to providemagnesium as a stabilizing agent in the compositions of the presentinvention, the molar ratio of magnesium chloride to citrate ranges fromabout 1:2 to about 1:10. In certain embodiments the ratio of magnesiumranges from about 1:1 to about 1:5. Preferably the molar ratio ofmagnesium chloride to citrate ranges from about 1:3 to about 1:5. Incertain embodiments, the concentration of magnesium ranges from about 1about 15 mM. In certain embodiments, the concentration of magnesiumranges from about 1 about 5 mM, about 5 to about 10 mM or about 10 toabout 15 mM. In certain embodiments, the concentration of magnesium isabout 2.5, about 5, about 7.5 or about 10 mM.

Another stabilizing agent which may be used in compositions of thepresent invention is a chloride-containing compound, such as sodiumchloride, which has molecular formula NaCl and molecular weight of58.44. Sodium chloride is used in some currently available formulationsof rapid acting insulin analogs, such as APIDRA® (insulin glulisine),which comprises 5 mg/mL sodium chloride and NOVOLOG® (insulin aspart),which comprises 0.58 mg/mL sodium chloride. In certain embodiments ofthe present invention wherein sodium chloride is used as a stabilizingagent, the concentration of sodium chloride ranges from about 1 to about50 mM. In certain embodiments of the present invention wherein sodiumchloride is used as a stabilizing agent, the concentration of sodiumchloride ranges from about 10 to about 40 mM. In certain embodiments,the concentration of sodium chloride ranges from about 15 to about 25mM. In certain embodiments, the concentration of sodium chloride isabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19 or about 20 mM.

Both MgCl₂ and NaCl result in the addition of chloride (Cl⁻) ions, andif the total chloride content of the composition is too high, theinsulin in the composition may crystallize at low temperatures and mayalso lead to instability at high temperatures. Thus, if MgCl₂ and/orNaCl are used as stabilizing agents, the total chloride content of thecomposition must be taken into consideration. Determining the totalchloride amount present in the composition if MgCl₂ and/or NaCl are usedas stabilizing agents must also take into consideration the fact thatchloride ions may also be added to the composition through the additionof other components, for example with the insulin bulk activepharmaceutical ingredient (API), through the addition of small amountsof HCl which may be necessary for pH adjustments, and/or in connectionwith the provision of Zn, which may be added in the form of a solutionprepared by solubilizing zinc oxide (ZnO) with HCl. Thus, the totalchloride concentration from all sources must be considered if MgCl₂and/or NaCl are to be used as stabilizing agents. In terms ofconcentrations, low temperature insulin crystallization has beenobserved in compositions containing about 100 mM NaCl, but such issueswere not observed in compositions containing up to about 30 mM totalchloride. In addition, the low temperature crystallization issuesassociated with relatively high chloride concentrations have also beenfound to be sensitive to citrate concentrations. Thus, compositions ofthe present invention having citrate concentrations at the lower end ofthe range provided for herein may be more tolerant of relatively higherchloride concentrations than compositions having citrate concentrationsat the higher end of the range provided for herein. For example, theaddition of sodium chloride concentrations as high as 50-75 mM toformulations containing 25 mM citrate have been observed to lead to lowtemperature crystallization issues, but such issues are not consistentlyobserved either when 50 mM sodium chloride is added to a 15 mM citrateformulation or when 25 mM sodium chloride is added to a 25 mM citrateformulation. The total chloride added through the use of NaCl and/orMgCl₂ as stabilizing agents should not be more than about 50 mM. Incertain embodiments of the present invention, the total chlorideconcentration, from all sources, ranges from about 10 to about 50 mM. Incertain embodiments, the total chloride concentration ranges from about13 to about 45 mM. In certain embodiments, the total chlorideconcentration ranges from about 20 to about 25 mM. In certainembodiments, the total chloride concentration ranges from about 15 toabout 35 mM. In certain embodiments, the total chloride concentrationranges from about 20 to about 25 mM. In certain embodiments, the totalchloride concentration is about 15 mM, about 16 mM, about 17 mM, about18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM,about 24 mM or about 25 mM.

In certain embodiments, the composition may include more than oneadditional stabilizing agent in order to ensure the compositionmaintains a commercially acceptable stability profile. A preferredcombination of stabilizing agents which may be used in compositions ofthe present invention includes excess zinc and magnesium. Anotherpreferred combination of stabilizing agents which may be used incompositions of the present invention includes zinc, a surfactant, suchas poloxamer 188, and magnesium chloride or sodium chloride.

The compositions of the present invention include one or morepreservatives, which provide anti-microbial properties and may furtherprovide stability benefits. The compositions are sterile when firstproduced, however, when the composition is provided in a multi-use vialor cartridge, an anti-microbial preservative compound or mixture ofcompounds that is compatible with the other components of theformulation is typically added at sufficient strength to meet regulatoryand pharmacopoeial anti-microbial preservative requirements. See U.S.Pharmacopeia Monographs. Insulin lispro injection. USP29-NF24; BritishPharmacopeia Monographs 2008 Volume III: Insulin aspart injection; U.S.Pharmacopeia Monographs. Insulin assays; and U.S. Pharmacopeia generalchapters. USP29-NF24. Rockville, Md.: U.S. Pharmacopeial Convention;2005. Antimicrobial effectiveness testing; pp. 2499-2500. Preferredpreservatives are aryl acids and phenolic compounds, or mixtures of suchcompounds. Effective concentrations can be ascertained readily using themethods referenced above. Preservatives commonly used in insulinproducts include phenol (CAS No. 108-95-2, molecular formula C₆H₅OH,molecular weight 94.11), and m-cresol (CAS No. 108-39-4, molecularformula C₇H₈O, molecular weight 108.14). Present commercialcompositions, for example, contain 3.15 mg/mL m-cresol (HUMALOG® andAPIDRA®), 1.72 mg/mL m-cresol and 1.50 mg/mL phenol (NOVOLOG®), and 2.5mg/mL m-cresol (HUMULIN® R U-500). In an embodiment, the preservative isselected from the group consisting of phenol and m-cresol. Preferablythe preservative is m-cresol. In certain embodiments the m-cresolconcentration is from about 2.5 to about 3.8 mg/mL. Preferably theconcentration of m-cresol is about 3.15 mg/mL.

It is desirable to approximately match the tonicity (i.e., osmolality)of body fluids at the injection site as closely as possible whenadministering the compositions because solutions that are notapproximately isotonic with body fluids can produce a painful stingingsensation when administered. Thus, it is desirable that the compositionsbe approximately isotonic with body fluids at the sites of injection. Ifthe osmolality of a composition in the absence of a tonicity agent issufficiently less than the osmolality of the tissue (for blood, about300 mOsmol/kg; the European Pharmacopeial requirement for osmolalityis >240 mOsmol/kg), then a tonicity agent should generally be added toraise the tonicity of the composition to about 300 mOsmol/kg. Theosmolality of the composition is determined by the identities andconcentrations of other excipients in the composition, including thestabilizing agent(s). Thus, the concentrations of all of the variousexcipients in a composition must be assessed in order to determinewhether a tonicity agent must be added and such assessments anddeterminations are readily made using standard techniques. SeeRemington: The Science and Practice of Pharmacy, David B. Troy and PaulBeringer, eds., Lippincott Williams & Wilkins, 2006, pp. 257-259;Remington: Essentials of Pharmaceutics, Linda Ed Felton, PharmaceuticalPress, 2013, pp. 277-300. Typical tonicity agents include glycerol(glycerin), mannitol and sodium chloride. If the addition of a tonicityagent is required, glycerol is preferred. In certain embodiments theconcentration of glycerol is from about 1 to about 16 mg/mL. In certainembodiments, the concentration of glycerol is from about 1 to about 2mg/mL, about 3 to about 4 mg/mL, about 5 to about 6 mg/mL, about 7 toabout 8 mg/mL, about 9 to about 10 mg/mL, about 11 to about 12 mg/mL,about 13 to about 14 mg/mL, or about 15 to about 16 mg/mL. In certainembodiments, the concentration of glycerol is about 5, about 12 or about16 mg/mL.

Citrate, which as noted above is added to contribute to improvements intime action, is also known to also have buffering properties, but ifdesired an additional buffering compound may be included. Examples ofsuch buffering compounds are phosphate buffers, such as dibasic sodiumphosphate, sodium acetate and tris(hydroxymethyl)aminomethane, or TRIS.If an additional buffering compound is necessary, TRIS or phosphatebuffers are preferred. The pH for commercial insulin compositions isusually in the range of 7.2 to 7.6, with 7.4±0.1 as a common target pH.The pH of the present invention is typically from about 7.0 to about 7.8and it is adjusted using physiologically appropriate acids and bases,typically hydrochloric acid 10% and sodium hydroxide 10%. Preferably,the pH is about 7.4.

The route of administration for the compositions of the presentinvention will typically be by self-administered subcutaneous injection,e.g., by use of a syringe or a pen device, or by continuous subcutaneousinsulin infusion therapy with an insulin pump device, thoughintravenous, intradermal, or intraperitoneal routes may also be used.

As noted above, the present invention also provides an article ofmanufacture comprising a pharmaceutical composition. In certainembodiments, the article of manufacture is a multi-use vial. In otherembodiments, the article of manufacture is a multi-use pre-filledcartridge. In other embodiments, the article of manufacture is are-usable pen injector. In other embodiments, the article of manufactureis a disposable pen device. In other embodiments, the article ofmanufacture is a pump device for continuous subcutaneous insulininfusion therapy. In other embodiments, the article of manufacture is acontainer closure system for use in a pump device for continuoussubcutaneous insulin infusion therapy.

In an embodiment, the present invention provides a pharmaceuticalcomposition comprising: an insulin; citrate, in a concentration fromabout 5 to about 25 mM; treprostinil, in a concentration from about 0.04to about 20 μg/mL; zinc, in a concentration sufficient to provide atleast 2 zinc ions per six molecules of insulin; a preservative; and oneor more additional stabilizing agents; and having a pH of about 7.0 toabout 7.8 at room temperature.

In certain embodiments, the concentration of zinc ranges from about 0.2to about 2 mM. In certain embodiments, the zinc concentration is fromabout 0.2 to about 1 mM. In certain embodiments, the zinc concentrationis from about 0.6 to about 0.8 mM. In certain embodiments, the zincconcentration is about 0.6 mM. In certain embodiments, the zincconcentration is about 0.7 mM. In certain embodiments, the zincconcentration is about 0.8 mM. In certain embodiments, the zincconcentration is about 0.9 mM.

In certain embodiments, the one or more additional stabilizing agentsare selected from the group consisting of a surfactant, magnesiumchloride and sodium chloride.

In certain embodiments, the one or more additional stabilizing agentscomprise a surfactant, which is present in a concentration of about0.003 to about 2% w/v. In certain embodiments, the surfactant ispoloxamer 188. In certain embodiments, the concentration of poloxamer188 is from about 0.06 to about 0.12% w/v. In certain embodiments, theconcentration of poloxamer 188 is about 0.06% w/v. In certainembodiments, the concentration of poloxamer 188 is about 0.09% w/v. Incertain embodiments, the concentration of poloxamer 188 is about 0.12%w/v.

In certain embodiments, the one or more additional stabilizing agentscomprise magnesium chloride, which is present in a concentrationresulting in a molar ratio of magnesium chloride to citrate from about1:2 to about 1:10. In certain embodiments, the ratio of magnesium rangesfrom about 1:1 to about 1:5. In certain embodiments, the molar ratio ofmagnesium chloride to citrate is from about 1:3 to about 1:5. In certainembodiments, the concentration of magnesium ranges from about 1 about 15mM. In certain embodiments, the concentration of magnesium ranges fromabout 1 about 5 mM, about 5 to about 10 mM or about 10 to about 15 mM.In certain embodiments, the concentration of magnesium is about 2.5,about 5, about 7.5 or about 10 mM.

In certain embodiments, the one or more additional stabilizing agentscomprise sodium chloride, which is present in a concentration of about10 to about 40 mM. In certain embodiments, the sodium chlorideconcentration is from about 15 to about 25 mM. In certain embodiments,the total chloride concentration is not more than about 50 mM. Incertain embodiments, the total chloride concentration ranges from about10 to about 50 mM. In certain embodiments, the total chlorideconcentration ranges from about 13 to about 45 mM. In certainembodiments, the total chloride concentration ranges from about 20 toabout 25 mM. In certain embodiments, the total chloride concentrationranges from about 15 to about 35 mM. In certain embodiments, the totalchloride concentration ranges from about 20 to about 25 mM. In certainembodiments, the total chloride concentration is about 15 mM, about 16mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM,about 22 mM, about 23 mM, about 24 mM or about 25 mM.

In certain embodiments, the insulin concentration is from about 100 toabout 500 U/mL. In certain embodiments, the insulin concentration isfrom about 100 to about 300 U/mL. In certain embodiments, the insulinconcentration is either about 100 U/mL or about 200 U/mL. In certainembodiments, the insulin is insulin lispro.

In certain embodiments, the concentration of citrate is from about 10 toabout 25 mM. In certain embodiments, the concentration of citrate isabout 15 mM.

In certain embodiments, the composition comprises treprostinil in aconcentration from about 0.04 to about 20 μg/mL. In certain embodiments,the concentration of treprostinil is from about 0.04 to about 10 μg/mL.In certain embodiments, the concentration of treprostinil is from about0.5 to about 2 μg/mL. In certain embodiments, the concentration oftreprostinil is about 1 μg/mL.

In certain embodiments, the preservative is m-cresol. In certainembodiments, the concentration of m-cresol is from about 2.5 to about3.8 mg/mL. In certain embodiments, the concentration of m-cresol isabout 3.15 mg/mL.

In certain embodiments, the composition further comprises a tonicityagent. In certain embodiments, the tonicity agent is glycerol. Incertain embodiments, the concentration of glycerol is from about 1 toabout 15 mg/mL. In certain embodiments, the concentration of glycerol isfrom about 1 to about 2 mg/mL.

In certain embodiments, the pharmaceutical composition provides for anuptake of insulin into the blood, onset of action and/or duration ofaction that is at least 20% more rapid than for compositions whichcontain the same insulin but which do not contain citrate ortreprostinil, when measured by one or more pharmacokinetic orpharmacodynamic parameters relevant to time action, such as: time tomaximum insulin concentration (Tmax); time to reach one half of themaximum insulin concentration (early ½ Tmax); time to reach one half ofthe maximum insulin concentration during the declining phase of theconcentration-over-time curve (late 12 Tmax); time between early andlate ½ Tmax (Tmax spread); percentage of total insulin dose absorbed atdifferent times based on fractional area under the insulin concentrationcurve (e.g., AUC_(0-30min), AUC_(0-60min), AUC_(0-120 min),AUC_(0-180min)); time to reach one half of the total insulinconcentration (T50); time to reach maximal glucose infusion rate(GIRmax), time to reach one half of the maximum glucose infusion rate(early ½ GIRmax); time to reach one half of the maximum glucose infusionrate during the declining phase of the concentration-over-time curve(late ½ GIRmax); percentage of total glucose infused at different timesbased on fractional area under the GIR curve (e.g., GIR_(0-30min),GIR_(0-60min), GIR_(0-120 min), GIR_(0-180min)).

In certain embodiments, the pharmaceutical composition provides for anuptake of insulin into the blood, onset of action and/or duration ofaction that is at least 30%, at least 40% or at least 50% more rapidthan for compositions which contain the same insulin but which do notcontain citrate or treprostinil, when measured by one or morepharmacokinetic or pharmacodynamic parameters described above.

In certain embodiments, the pharmaceutical composition provides for anuptake of insulin into the blood, onset of action and/or duration ofaction that is between about 20 to about 50%, between about 20 to about30%, between about 30 to about 40% or between about 40 to about 50% morerapid than for compositions which contain the same insulin but which donot contain citrate or treprostinil, when measured by one or morepharmacokinetic or pharmacodynamic parameters described above.

In certain embodiments, the pharmaceutical composition does not includeany additional chelating agent, such as EDTA, any additionalvasodilatory agent, such as nitroglycerin, and/or any oligosaccharides.

In certain embodiments, the pharmaceutical composition is stable toallow for storage of at least 24 months at 2-8° C. and up to 28 daysin-use at temperatures of up to 30° C. for vials or cartridges inre-usable pen injectors. In certain embodiments, the pharmaceuticalcomposition is stable to allow for storage of at least 36 months at 2-8°C. and up to 28 days in-use at temperatures of up to 30° C. for vials orcartridges in re-usable pen injectors.

In certain embodiments, the composition is stable to allow for use in apump device for continuous subcutaneous insulin infusion therapy for upto 7 days.

Additional embodiments of the present invention include those describedbelow:

1. A pharmaceutical composition comprising: an insulin; citrate; andtreprostinil.

2. The pharmaceutical composition of the above-described embodiment,wherein the insulin is selected from the group consisting of humaninsulin, insulin lispro, insulin aspart or insulin glulisine.

3. The pharmaceutical composition of any of the above-describedembodiments, wherein the insulin is insulin lispro.

4. The pharmaceutical composition of any of the above-describedembodiments, wherein the insulin concentration is from about 100 toabout 500 U/mL.

5. The pharmaceutical composition of any of the above-describedembodiments, wherein the insulin concentration is from about 100 toabout 300 U/mL.

6. The pharmaceutical composition of any of the above-describedembodiments, wherein the insulin concentration is from about 100 toabout 200 U/mL.

7. The pharmaceutical composition of any of the above-describedembodiments, wherein the insulin concentration is about 100 U/mL.

8. The pharmaceutical composition of any of the above-describedembodiments, wherein the insulin concentration is about 200 U/mL.

9. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of citrate is from about 5 toabout 25 mM.

10. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of citrate is from about 15 toabout 25 mM.

11. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of citrate is from about 15 toabout 20 mM.

12. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of citrate is about 5, about 10,about 12, about 15, about 18, about 20 or about 25 mM.

13. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of citrate is about 15 mM

14. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of treprostinil is from about0.04 to about 20 μg/mL.

15. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of treprostinil is from about0.04 to about 10 μg/mL.

16. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of treprostinil is from about 0.5to about 10 μg/mL.

17. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of treprostinil is from about 0.5to about 2 μg/mL.

18. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of treprostinil is about 0.5,about 0.6, about 1, about 2, about 2.3, about 9.3 or about 10 μg/mL.

19. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of treprostinil is about 1 μg/mL.

20. The pharmaceutical composition of any of the above-describedembodiments, wherein the composition further comprises zinc.

21. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is from about 0.2 toabout 2 mM.

22. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is from about 0.3 toabout 1.7 mM.

23. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is from about 0.7 toabout 1.7 mM.

24. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is from about 0.3 toabout 1 mM.

25. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is from about 0.4 toabout 0.8 mM.

26. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is about 0.3, about 0.4,about 0.5, about 0.6, about 0.7 about 0.8, about 0.9 mM, about 1.25 orabout 1.7 mM.

27. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is about 0.6 mM.

28. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is about 0.7 mM.

29. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is about 0.8 mM.

30. The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is about 0.9 mM.

31. The pharmaceutical composition of any of the above-describedembodiments, further comprising magnesium.

32. The pharmaceutical composition of any of the above-describedembodiments comprising magnesium which is present in a concentrationresulting in a molar ratio of magnesium to citrate from about 1:2 toabout 1:10.

33. The pharmaceutical composition of any of the above-describedembodiments comprising magnesium which is present in a concentrationresulting in a molar ratio of magnesium to citrate from about 1:1 toabout 1:5.

34. The pharmaceutical composition of the above-described embodiments,wherein the molar ratio of magnesium to citrate is from about 1:3 toabout 1:5.

35. The pharmaceutical composition of any of the above-describedembodiments, comprising magnesium in a concentration from about 1 toabout 15 mM.

36. The pharmaceutical composition of any of the above-describedembodiments, comprising magnesium in a concentration from about 2.5 toabout 10 mM.

37. The pharmaceutical composition of any of the above-describedembodiments, comprising magnesium in a concentration from about 5 toabout 10 mM.

38. The pharmaceutical composition of any of the above-describedembodiments, comprising magnesium in a concentration from about 2.5 toabout 7.5 mM.

39. The pharmaceutical composition of any of the above-describedembodiments, comprising magnesium in a concentration of about 2.5, about5, about 7.5 or about 10 mM.

40. The pharmaceutical composition of any of the above-describedembodiments comprising magnesium wherein the magnesium is provided asmagnesium chloride.

41. The pharmaceutical composition of the above-described embodiments,further comprising sodium chloride.

42. The pharmaceutical composition of any of the above-describedembodiments, wherein the composition comprises sodium chloride in aconcentration ranging from about 1 to about 50 mM.

43. The pharmaceutical composition of any of the above-describedembodiments, wherein the composition comprises sodium chloride in aconcentration ranging from about 10 to about 40 mM.

44. The pharmaceutical composition of any of the above-describedembodiments, wherein the composition comprises sodium chloride in aconcentration ranging from about 15 to about 25 mM.

45. The pharmaceutical composition of any of the above-describedembodiments, wherein the composition comprises sodium chloride in aconcentration ranging from about 1 to about 20 mM.

46. The pharmaceutical composition of any of the above-describedembodiments, wherein the composition comprises sodium chloride in aconcentration of about 1, about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11, about 12, about 13, about14, about 15, about 16, about 17, about 18, about 19 or about 20 mM.

47. The pharmaceutical composition of any of the above-describedembodiments, wherein the total chloride concentration is from about 1 toabout 100 mM.

48. The pharmaceutical composition of any of the above-describedembodiments, wherein the total chloride concentration is from about 10to about 100 mM.

49. The pharmaceutical composition of any of the above-describedembodiments, wherein the total chloride concentration ranges from about10 to about 50 mM.

50. The pharmaceutical composition of any of the above-describedembodiments, wherein the total chloride concentration ranges from about13 to about 45 mM.

51. The pharmaceutical composition of any of the above-describedembodiments, wherein the total chloride concentration ranges from about16 to about 35 mM.

52. The pharmaceutical composition of any of the above-describedembodiments, wherein the total chloride concentration ranges from about20 to about 25 mM.

53. In certain embodiments, the total chloride concentration is about 15mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM,about 21 mM, about 22 mM, about 23 mM, about 24 mM or about 25 mM.

54. The pharmaceutical composition of any of the above-describedembodiments, wherein the composition further comprises a surfactant.

55. The pharmaceutical composition of any of the above-describedembodiments, wherein the composition further comprises poloxamer 188.

56. The pharmaceutical composition of any of the above-describedembodiments, comprising poloxamer 188 in a concentration of about 0.003to about 2% w/v.

57. The pharmaceutical composition of any of the above-describedembodiments, comprising poloxamer 188 in a concentration of about 0.003to about 0.3% w/v.

58. The pharmaceutical composition of any of the above-describedembodiments, comprising poloxamer 188 in a concentration of about 0.01to about 0.2% w/v.

59. The pharmaceutical composition of any of the above-describedembodiments, comprising poloxamer 188 in a concentration of about 0.06to about 0.12 w/v.

60. The pharmaceutical composition of any of the above-describedembodiments, wherein the composition further comprises a preservative.

61. The pharmaceutical composition of any of the above-describedembodiments, comprising a preservative which is selected from the groupconsisting of m-cresol and phenol.

62. The pharmaceutical composition of any of the above-describedembodiments, comprising a preservative which is m-cresol.

63. The pharmaceutical composition of any of the above-describedembodiments, comprising m-cresol in a concentration from about 2.5 toabout 3.8 mg/mL.

64. The pharmaceutical composition of any of the above-describedembodiments, comprising m-cresol in a concentration of about 3.15 mg/mL.

65. The pharmaceutical composition of the above-described embodiments,further comprising a tonicity agent.

66. The pharmaceutical composition of the above-described embodiments,comprising a tonicity agent which is glycerol.

67. The pharmaceutical composition of the above-described embodiments,comprising glycerol in a concentration from about 1 to about 20 mg/mL.

68. The pharmaceutical composition of the above-described embodiments,comprising glycerol in a concentration from about 1 to about 15 mg/mL.

69. The pharmaceutical composition of the above-described embodiments,comprising glycerol in a concentration from about 1 to about 2 mg/mL,about 3 to about 4 mg/mL, about 5 to about 6 mg/mL, about 7 to about 8mg/mL, about 9 to about 10 mg/mL, about 11 to about 12 mg/mL, about 13to about 14 mg/mL or about 15 to about 16 mg/mL.

70. The pharmaceutical composition of the above-described embodiments,comprising glycerol in a concentration of about 5, about 12 or about 16mg/mL.

71. The pharmaceutical composition of the above-described embodiments,comprising glycerol in a concentration of about 12 mg/mL.

The invention is further illustrated by the following examples, whichare not to be construed as limiting.

EXAMPLES

Pharmacokinetic and Pharmacodynamic Studies

Insulin Lispro Formulated with 0.5 μg/ml Treprostinil and/or 15 mMSodium Citrate

Diabetic (Alloxan induced), castrated, male Yucatan miniature swine(average age 23 months, average body weight 45 kgs) with previouslyfitted vascular access ports are used under the supervision of staff andveterinarians. The diabetic animals are housed individually and haveaccess to fresh water at all times. They are fed two meals per day of astandard diet and receive appropriate maintenance basal and prandialinsulin twice per day to manage their diabetic condition.

Test articles (Compositions A, B and C in the table below) areformulated by adding the indicated amounts of citrate and/ortreprostinil to a vial of Humalog®. The necessary quantity oftreprostinil is withdrawn from a vial of 1 mg/mL Remodulin®. Each mL ofRemodulin® also contains 3 mg m-cresol, 6.3 mg sodium citrate and 5.3 mgsodium chloride, so the addition of treprostinil to the testcompositions also results in the addition of small amounts of theseexcipients, as well as a slight dilution of the Humalog® excipients.Such small quantities are not believed to affect the properties of thecompositions, however, and are thus not reflected in Table 1 below.

TABLE 1 Name Formulation Composition A 100 Units/ml insulin lispro 0.5μg/ml treprostinil 1.88 mg/ml phosphate 3.15 mg/ml m-cresol 16 mg/mlglycerol 0.3 mM Zn pH 7.4 Composition B 98 Units/ml insulin lispro 15 mMsodium citrate 1.88 mg/ml phosphate 3.15 mg/ml m-cresol 16 mg/mlglycerol 0.3 mM Zn pH 7.4 Composition C 98 Units/ml insulin lispro 15 mMsodium citrate 0.5 μg/ml treprostinil 1.88 mg/ml phosphate 3.15 mg/mlm-cresol 16 mg/ml glycerol 0.3 mM Zn pH 7.4 Humalog ® 100 U/mL insulinlispro 1.88 mg/ml phosphate 16 mg/mL glycerol 3.15 mg/mL meta-cresol 0.3mM zinc pH 7.4

The day prior to study, animals are fed half their daily ration andreceive 0.2 U/kg Humalog® Mix 75/25 Insulin at their morning maintenanceadministration. All study animals are food-fasted overnight and do notreceive their evening insulin or meal prior to drug administration onstudy day.

On the morning of study, all animals are placed into slings forrestraint and have their vascular access ports accessed (equipped forblood sampling) and checked for patency. The animals are randomly placedinto treatment groups.

Study is a full crossover design with n=20. One animal is withheld fromtwo of the four parts due to port non-patency, so compositions A and Care n=19 while composition B and Humalog® are n=20.

After two baseline blood samples are collected (−30 and −20 min), theanimals are returned to their pens and are fed ˜300 g. Twenty minutesafter the presentation of the fully consumed meal, the animals areinjected with test article subcutaneously in the flank (0 min) with aTerumo insulin syringe (0.3 or 0.5 ml with ½″ needle). All study animalshave access to clean, fresh water throughout the remaining bloodcollection period.

Serial blood samples (2.0 mL each) are collected from each animal at thefollowing time points: −30, −20 (then immediately fed), 0 (just beforedose), 5, 10, 15, 30, 45, 60, 75, 90, 105, 120, 150, 180, 240, and 360minutes following the SC dosing.

Blood samples (anticoagulant: none [serum]) are maintained at ambienttemperature for at least 30 minutes but no more than 2 hours to allowfor clotting. Serum is then separated by centrifugation and divided intotwo aliquots and stored frozen at approximately −70° C.

Serum glucose concentrations are determined using an automated AU480Clinical Chemistry Analyzer (Beckman Coulter, Inc., Brea, Calif.).Aliquot for PK is shipped to EMD Millipore Corp., St. Charles, Mo. ondry ice by a next day shipping service and included a detailed samplemanifest.

Serum glucose data are represented in Table 2 below as mean(mg/dL)+/−standard error of the mean (SEM) unless otherwise specified.

TABLE 2 Composition Composition Composition Time Humalog ® A B C (min)AVG SEM AVG SEM AVG SEM AVG SEM −30 290.2 8.6 286.8 9.0 289.2 8.1 283.28.4 −20 297.6 8.5 299.2 10.2 297.6 9.0 297.5 7.5 0 311.3 8.6 311.0 10.7312.2 9.5 309.6 7.4 5 318.1 8.9 316.8 10.0 319.0 9.6 314.9 8.1 10 318.98.6 315.3 11.4 305.2 11.7 295.7 10.3 15 312.3 9.2 297.6 13.5 276.6 12.8265.1 11.6 30 297.1 9.8 251.9 18.6 220.3 16.2 205.5 17.0 45 288.3 12.0233.1 16.7 204.5 18.5 209.8 17.1 60 244.1 14.7 187.9 17.5 178.5 18.6159.2 20.0 75 231.5 15.3 166.6 18.2 169.0 20.4 146.3 20.4 90 204.6 15.6145.7 18.5 157.7 21.4 133.2 21.1 105 181.9 15.9 127.0 18.1 144.7 21.4118.7 18.9 120 153.3 16.2 111.4 17.3 128.2 21.1 111.7 18.5 150 120.216.8 86.4 15.8 115.7 20.0 102.9 15.9 180 101.5 16.5 76.4 14.5 106.3 18.297.0 13.2 240 82.8 12.2 92.3 14.2 100.7 13.3 98.6 12.3 360 90.3 14.4130.5 18.2 111.8 17.2 126.3 17.0

Serum insulin concentrations for PK analysis are measured using a totalinsulin RIA. Lower and upper limits of quantitation for the assay are 20pM and 5000 pM, respectively. Values below the lower limit ofquantitation are assumed to be 20 pM. Non-compartmental pharmacokineticanalyses are performed using Phoenix WinNonlin v6.3. PK parametersderived from serum insulin concentrations are provided in Table 3 below.

TABLE 3 Tmax Early ½ Max Late ½ Max Tmax Spread T50 Composition (min)(min) (min) (min) (min) Humalog ® Mean (SE) 71.3 (7.3) 29.6 (4.1) 175(14) 145 (14) 127 (6) A Mean (SE) 74.2 (7.6) 24.1 (3.8) 146 (12) 122(13) 102 (5) B Mean (SE) 55.5 (7.8) 16.5 (4.9) 130 (12) 113 (14) 100 (6)C Mean (SE) 42.1 (6.6) 15.9 (3.9) 98.5 (13.4) 82.6 (13.6) 83.4 (5.7)

This study supports that compositions of the present invention haveimproved pharmacokinetic and/or pharmacodynamic time action.

Insulin Lispro Formulated with 10 mM Citrate and/or 10 mg/mLTreprostinil

A study on compositions comprising varying concentrations of citrateand/or μg/mL treprostinil is performed in diabetic (Alloxan induced),castrated, male Yucatan miniature swine (average age 24 months, averagebody weight 43 kgs) following generally the procedures described above.

Test articles (Compositions D-H in the table below) are formulated byadding the indicated amounts of citrate and/or treprostinil to a vial ofHumalog®. As with the study described above, the necessary quantity oftreprostinil is withdrawn from a vial of 1 mg/mL Remodulin®, so theaddition of treprostinil to the test compositions also results in theaddition of small quantities of m-cresol, sodium citrate and sodiumchloride, as well as a slight dilution of the Humalog® excipients. Suchsmall quantities are not believed to affect the properties of thecompositions, however, and are thus not reflected in Table 4 below.

TABLE 4 Name Formulation Composition Composition D 99.7 U/ml insulinlispro 5 mM citrate 0.3 mM zinc 3.15 mg/mL meta-cresol 16 mg/mlglycerine 1.88 mg/ml phosphate Composition E 99.2 U/ml insulin lispro 10mM citrate 0.3 mM zinc 3.15 mg/mL meta-cresol 16 mg/ml glycerine 1.88mg/ml phosphate Composition F 99.2 U/ml insulin lispro 10 μg/mltreprostinil 0.3 mM zinc 3.15 mg/mL meta-cresol 16 mg/ml glycerine 1.88mg/ml phosphate Composition G 100.2 U/ml Insulin lispro 5 mM citrate 10μg/ml treprostinil 0.3 mM zinc 3.15 mg/mL meta-cresol 16 mg/ml glycerine1.88 mg/ml phosphate Composition H 99.6 U/ml Insulin lispro 10 mMcitrate 10 μg/ml treprostinil 0.3 mM zinc 3.15 mg/mL meta-cresol 16mg/ml glycerine 1.88 mg/ml phosphate Humalog ® 100 U/mL KPB 1.88 mg/mLdibasic sodium phosphate 16 mg/mL glycerol 3.15 mg/mL meta-cresol 0.3 mMzinc pH 7.4

Study is designed as a 19 pig full cross over design in which each pigis studied on each test article. A few animals do not participate onstudy due to port non-patency therefore formulations D and E are n=18while formulations F-H are n=19 and Humalog® n=17.

Serum glucose data are represented in Table 5 below as mean(mg/dL)+/−SEM) unless otherwise specified.

TABLE 5 Composition Composition Composition Composition Composition TimeHumalog ® D E F G H (min) AVG SEM AVG SEM AVG SEM AVG SEM AVG SEM AVGSEM −30 340.5 8.2 340.2 8.9 321.2 7.3 335.2 8.5 337.4 7.5 331.8 10.1 −20344.4 8.0 341.3 9.2 326.2 7.8 339.8 7.7 344.6 6.7 338.6 11.0 0 362.6 7.7353.2 10.3 339.4 9.5 357.8 7.9 354.7 7.9 349.1 11.4 5 377.6 7.9 370.69.8 349.5 11.9 367.9 8.0 369.1 8.5 368.2 12.4 10 376.7 8.4 360.7 9.3329.6 12.0 361.0 11.1 361.9 9.0 345.6 13.4 15 372.4 10.1 341.7 13.2307.6 13.7 348.1 12.3 342.6 9.1 321.6 14.6 30 339.8 17.9 292.2 17.3256.6 18.5 304.1 14.7 299.0 12.9 276.4 20.6 45 301.5 21.8 251.1 21.0230.9 20.9 263.1 18.0 254.6 16.0 237.3 24.2 60 272.4 25.7 224.2 21.3207.3 20.1 230.9 21.6 230.3 18.3 208.8 26.4 75 253.0 26.7 202.3 22.6178.2 19.3 197.2 22.8 196.9 20.3 168.5 25.2 90 220.2 28.3 186.1 23.0164.6 20.5 173.4 23.5 173.6 21.4 153.8 26.2 105 195.6 27.6 148.2 20.0144.3 19.6 148.6 23.1 147.6 20.9 131.8 24.2 120 184.4 27.5 133.3 19.1129.9 18.2 130.0 22.6 124.9 20.4 115.4 23.0 150 158.2 24.8 114.1 17.7104.6 18.5 101.4 18.6 98.8 16.8 90.1 18.3 180 134.3 22.0 102.7 16.5 86.115.8 90.8 17.5 92.2 14.6 76.8 15.9 240 122.4 19.7 93.7 16.4 75.2 12.493.5 15.0 91.1 17.5 74.4 11.7 360 118.4 17.1 104.0 15.8 90.7 15.1 113.913.7 113.7 14.1 105.1 11.1

Serum insulin concentrations and PK parameters for Humalog® control andcompositions E, F and H are generated and analyzed generally asdescribed above, and PK results are provided in table 6 below.

TABLE 6 Tmax Early ½ Max Late ½ Max Spread T50 Composition (min) (min)(min) (min) (min) Humalog ® Mean (SE) 69.7 (12.5) 17.8 (4.1) 155 (16)138 (16) 113 (7) E Mean (SE) 53.8 (10.3) 6.46 (0.55) 151 (15) 144 (15)104 (5) F Mean (SE) 54.2 (7.29) 23.1 (4.8) 110 (11) 87.1 (10.8) 89.4(5.7) H Mean (SE) 39.5 (4.95) 9.85 (1.77) 109 (11) 99.6 (11.5) 90.7(5.5)

This study supports that compositions of the present invention haveimproved pharmacokinetic and/or pharmacodynamic time action.

Insulin Lispro Formulated with 15 mM Citrate and 0.6-9.3 μg/mLTreprostinil

A study on compositions comprising 15 mM citrate and/or varyingconcentrations of treprostinil is performed in diabetic (Alloxaninduced), castrated, male Yucatan miniature swine (average age 25months, average body weight 43 kgs) following generally the proceduresdescribed above.

Test articles (Compositions I-M in the table below) are formulated byadding the indicated amounts of citrate and/or treprostinil, as well asMgCl₂ and/or NaCl in the compositions specified below, to a vial ofHumalog®. As with the study described above, the necessary quantity oftreprostinil is withdrawn from a vial of 1 mg/mL Remodulin®, so theaddition of treprostinil to the test compositions also results in theaddition of small quantities of m-cresol, sodium citrate and sodiumchloride, as well as a slight dilution of the Humalog® excipients. Suchsmall quantities are not believed to affect the properties of thecompositions, however, and are thus not reflected in Table 7 below.

TABLE 7 Name Formulation Composition Composition I 99.1 U/ml Insulinlispro 15 mM citrate 5 mM MgCl2 15 mM NaCl 0.3 mM zinc 3.15 mg/mLmeta-cresol 1.88 mg/mL dibasic sodium phosphate 16 mg/ml glycerin,Composition J 99.8 U/ml Insulin lispro 9.30 μg/ml treprostinil 0.3 mMzinc 3.15 mg/mL meta-cresol 1.88 mg/mL dibasic sodium phosphate 16 mg/mlglycerin Composition K 99.6 U/ml Insulin lispro 15 mM citrate 5 mM MgCl215 mM NaCl 0.60 μg/ml treprostinil 0.3 mM zinc 3.15 mg/mL meta-cresol1.88 mg/mL dibasic sodium phosphate 16 mg/ml glycerin Composition L100.4 U/ml Insulin lispro 15 mM citrate 5 mM MgCl2 15 mM NaCl 2.30 μg/mltreprostinil 0.3 mM zinc 3.15 mg/mL meta-cresol 1.88 mg/mL dibasicsodium phosphate 16 mg/ml glycerin Composition M 100.3 U/ml Insulinlispro 15 mM citrate 5 mM MgCl2 15 mM NaCl 9.30 μg/ml treprostinil 0.3mM zinc 3.15 mg/mL meta-cresol 1.88 mg/mL dibasic sodium phosphate 16mg/ml glycerin Humalog ® 100 U/mL KPB 1.88 mg/mL dibasic sodiumphosphate 16 mg/mL glycerol 3.15 mg/mL meta-cresol 0.3 mM zinc pH 7.4

Study is designed as a 17 pig full cross over design in which each pigis studied on each test article. Some animals do not participate onstudy due to port non-patency or low baseline glucose. Data for Humalog®is n=17, Composition I is n=17, Composition J is n=16, Composition K isn=15, Composition L is n=16 and Composition M is n=16.

Serum glucose data are represented in Table 8 below as mean(mg/dL)+/−SEM unless otherwise specified.

TABLE 8 Composition Composition Composition Composition Composition TimeHumalog ® I J K L M (min) AVG SEM AVG SEM AVG SEM AVG SEM AVG SEM AVGSEM −30 302.5 7.0 324.0 7.2 322.4 11.0 319.6 10.4 337.6 13.6 321.6 7.9−20 310.7 7.8 330.7 7.6 330.3 10.3 324.5 12.1 345.3 13.8 325.4 7.6 0321.9 8.6 338.5 9.3 340.1 11.9 335.9 11.7 357.0 15.7 332.2 8.9 5 335.69.4 353.8 9.2 356.9 12.6 346.2 12.0 371.2 15.7 346.4 9.2 10 340.3 9.8347.0 9.6 356.7 11.7 337.7 14.4 365.6 16.5 339.2 11.8 15 326.6 9.7 315.610.9 340.1 10.7 306.5 13.7 339.0 17.3 302.4 13.4 30 276.6 17.6 264.117.9 271.9 15.5 240.0 15.4 274.7 23.1 244.0 20.6 45 241.2 22.9 220.220.2 237.5 14.4 197.9 20.8 226.7 26.8 195.1 22.6 60 202.1 26.0 186.921.5 192.9 17.8 160.3 21.4 195.9 27.5 157.9 23.6 75 182.5 27.2 159.922.1 155.7 19.8 140.9 21.2 171.4 28.1 136.8 22.7 90 158.0 27.4 141.223.9 133.0 19.2 119.5 21.5 153.2 27.4 122.0 21.1 105 142.7 25.2 127.123.2 118.0 19.8 105.3 19.8 132.6 25.6 115.9 20.3 120 122.8 23.6 115.821.6 98.7 18.5 94.5 17.3 121.8 24.8 99.3 18.6 150 107.9 21.6 101.3 18.272.5 15.7 82.8 14.3 110.3 21.7 87.6 17.1 180 90.4 17.1 98.6 18.2 66.411.9 79.2 11.2 101.8 20.4 88.2 15.4 240 83.4 14.5 96.5 16.8 72.0 8.097.1 11.8 111.2 22.2 98.3 14.3 360 86.0 18.6 96.1 13.3 122.2 16.5 149.519.8 156.8 21.2 125.4 20.4

Serum insulin concentrations and PK parameters are generated andanalyzed generally as described above, and PK results are provided intable 9 below.

TABLE 9 Early ½ Late ½ Tmax Max Max Spread T50 Composition (min) (min)(min) (min) (min) Humalog ® Mean 60.0 (7.6) 22.9 (5.8) 152 (17) 129 (18)107 (7) (SE) I Mean 57.1 (9.5) 16.0 (3.5) 144 (16) 128 (17) 102 (5) (SE)J Mean 55.3 (5.3) 13.7 (1.5) 139 (9) 125 (9) 96.8 (4.6) (SE) K Mean 55.0(6.2) 15.8 (2.2) 136 (10) 120 (11) 91.1 (4.8) (SE) L Mean 50.0 (7.2)13.2 (2.1) 121 (10) 108 (10) 90.7 (6.6) (SE) M Mean 52.5 (7.4) 11.7(1.5) 139 (18) 127 (18) 99.0 (7.4) (SE)

This study supports that compositions of the present invention haveimproved pharmacokinetic and/or pharmacodynamic time action.

Insulin Lispro Formulated with 10 μg/mL Treprostinil and Either 15 or 25mM Citrate

A study on compositions comprising 10 μg/mL treprostinil and either 15or 25 mM citrate is performed in diabetic (Alloxan induced), castrated,male Yucatan miniature swine (average age 33 months, average body weight49 kgs) following generally the procedures described above.

Test articles (Compositions N and O in the table below) are formulatedby adding the indicated amounts of citrate, treprostinil, NaCl and MgCl₂to a vial of Humalog®. As with the study described above, the necessaryquantity of treprostinil is withdrawn from a vial of 1 mg/mL Remodulin®,so the addition of treprostinil to the test compositions also results inthe addition of small quantities of m-cresol, sodium citrate and sodiumchloride, as well as a slight dilution of the Humalog® excipients. Suchsmall quantities are not believed to affect the properties of thecompositions, however, and are thus not reflected in Table 10 below.

TABLE 10 Name Formulation Composition Composition N 99.5 U/ml Insulinlispro 10 μg/ml treprostinil 15 mM citrate 5 mM MgCl₂ 15 mM NaCl 16mg/ml glycerin Composition O 99.6 U/ml insulin lispro 10 μg/mltreprostinil 25 mM citrate 5 mM MgCl₂ 15 mM NaCl 16 mg/ml glycerinHumalog ® 100 U/mL KPB 1.88 mg/mL dibasic sodium phosphate 16 mg/mLglycerol 3.15 mg/mL meta-cresol 0.3 mM zinc pH 7.4

Study is designed as a 20 pig full cross-over design in which each pigis studied on each test article. One animal does not participate inComposition N group due to port non-patency or low baseline glucose,data is n=19 for that group. Serum glucose data are represented in Table11 below as mean (mg/dL)+/−SEM unless otherwise specified.

TABLE 11 Time Humalog ® Composition N Composition O (min) AVG SEM AVGSEM AVG SEM −30 323.8 8.4 336.3 9.4 323.3 9.2 −20 332.6 9.1 344.7 9.5336.0 9.2 0 343.1 8.8 354.0 9.9 346.6 10.2 5 353.3 9.8 364.5 10.8 355.210.9 10 355.8 9.8 357.1 9.1 342.3 11.4 15 355.2 10.6 340.7 11.6 325.613.8 30 317.9 12.9 293.3 15.9 270.0 17.9 45 291.4 19.1 254.8 19.2 239.721.0 60 255.6 21.1 220.9 21.3 216.1 22.8 75 239.8 23.9 195.2 21.7 193.822.8 90 205.7 23.7 173.8 22.6 172.6 22.7 105 184.4 22.8 156.0 21.5 158.622.7 120 162.0 21.7 139.1 19.4 145.9 21.7 150 135.3 20.5 107.1 18.2116.8 19.4 180 119.2 18.4 86.1 16.5 104.8 18.3 240 84.1 13.3 83.3 13.180.8 14.8 360 63.4 11.4 108.6 14.2 86.1 10.8

Collectively, the studies described above support that the compositionsof the present invention have faster pharmacokinetic and/orpharmacodynamic action than commercial formulations of existing insulinanalog products.

Stability Studies

The stability of insulin lispro when formulated with a range of citrateconcentrations, treprostinil and other excipients is assessed. Thecompositions of exemplary formulations, shown in table 12 below, areprepared by formulating insulin lispro active pharmaceutical ingredientwith the other excipients indicated.

TABLE 12 Name Composition Composition P 100 U/mL insulin lispro 15 mMcitrate 10 μg/mL treprostinil 0.65 mM zinc 5 mM MgCl₂ 0.06% Poloxamer188 3.15 mg/mL m-cresol 17.5 mM Glycerol pH 7.4 Formulation Q 100 U/mLinsulin lispro 20 mM citrate 10 μg/mL treprostinil 0.65 mM zinc 6.6 mMMgCl₂ 0.06% Poloxamer 188 3.15 mg/mL m-cresol 15.68 mM Glycerol pH 7.4Formulation R 100 U/mL insulin lispro 25 mM citrate 10 μg/mLtreprostinil 8.3 mM MgCl₂ 0.65 mM zinc 0.06% Poloxamer 188 3.15 mg/mLm-cresol 13.82 mM Glycerol pH 7.4

The solutions are filtered using 50 ml Steriflip Vacuum filter with 0.22μM PES membrane (Cat #: SCGP00525, EMD Millipore, Billerica, Md.) anddistributed into either 10-mL vials with crimp-top or 3-mL glass insulincartridges, then incubated at 4° C. and 30° C. respectively. The 4° C.samples are stored without agitation for 64 days, while the 30° C.samples are incubated without agitation for 36 days, followed by asimulated in-use period of 28 days. In the in-use simulation thecontainer is inverted and a sample is drawn from each vial or cartridgethree times a day for 28 days. Two draws are done at each time point ondays before or after any Saturday, Sunday or holiday. Aliquots of 80 μLare drawn from each vial using Terumo U-100 insulin (½ cc 27 G×½″)syringe and 30 μL are drawn from each cartridge using the HumaPen LuxuraHD using the Comfort Point 6 mm (31 G×¼″) needle.

Samples are collected from day 36 (day 1 of the in-use period at 30°C.), day 50 and day 64 to be analyzed using reversed-phasehigh-performance liquid chromatography (RP-HPLC) and analytical sizeexclusion chromatography (SEC).

All formulations are also evaluated in 3 mL glass insulin cartridges at37° C. while shaking at ˜100 rpm. This condition is intended to beindicative of stability in continuous subcutaneous insulin infusiontherapy. Endpoint samples from this condition are analyzed using RP-HPLCand SEC.

RP-HPLC analysis is performed to assess protein purity in eachformulation at the stability time points using a UV detector at 214 nm.Each sample (5 μL) is separated using a Zorbax 300SB-C18, RapidResolution 4.6×150 mm 3.5-Micron column (Part #863973-902) at 40° C.with a flow rate of 0.6 mL/minute and mobile phase A (50 mM sulfate, pH2.3+20% acetonitrile (v/v)) and mobile phase B (50 mM sulfate, pH2.3+50% acetonitrile (v/v)). Gradient of mobile phase B at 0, 3, 15, 21,26, 27, 27.5 and 35.0 min is 21, 25, 25, 30, 80, 80, 21 and 21%,respectively. Insulin content is calculated using RP-HPLC by combiningthe integrations of the insulin main peak area and A21 area thendividing by an insulin lispro standard. Insulin loss for formulationsamples compared to Humalog® control is less than 5% for all samples outto 64 days at 4° C. and 30° C. Percentage of sample outside of main peakwith A21 is calculated by dividing chromatographic main peak area bytotal peak area and subtracting that area and A21 from 100. The percentoutside of the main peak area is less than 1.76% for all samples at 4°C. for 64 days and less than 2.48% for all samples at 30° C. for 64days.

For the SEC analysis, each sample (5 μL) is separated using a SepaxZenix-C SEC-80, 7.8×300 mm, 3 μm particles column (catalog #233080-7830)at 5° C. and a flow rate of 1.0 mL/minute with isocratic elution ofmobile phase (0.1% TFA, 50% ACN) over a run time of 25 minutes.

Percentage of high molecule weight polymer (% HMWP) is calculated byintegrating the total percent area of all peaks eluting prior to themain peak. Results (% HMWP) are given in the tables below. HMWPformation is less than 1% for all samples out to 64 days at 4° C. and30° C., and HMWP formation in citrate-containing samples is comparableto HMWP formation in Humalog® control samples.

TABLE 13 Vials stored at 4° C.: insulin content, units/mL. CompositionDay 36 Day 50 Day 64 P 100.07 99.89 99.36 Q 100.06 99.88 99.38 R 100.0399.85 99.29

TABLE 14 Vials stored at 4° C.: other related substances, percent.Composition Day 36 Day 50 Day 64 P 0.92 1.05 1.17 Q 0.93 1.07 1.12 R0.96 1.09 1.32

TABLE 15 Vials stored at 4° C.: high molecular weight species, percent.Composition Day 36 Day 50 Day 64 P 0.17 0.19 0.21 Q 0.16 0.20 0.22 R0.16 0.20 0.21

TABLE 16 Vials stored at 30° C.: insulin content, units/mL. CompositionDay 36 Day 50 Day 64 P 98.77 98.60 98.47 Q 98.67 98.50 98.46 R 98.8298.65 98.46

TABLE 17 Vials stored at 30° C.: other related substances, percent.Composition Day 36 Day 50 Day 64 P 1.23 1.40 1.53 Q 1.33 1.50 1.54 R1.18 1.35 1.54

TABLE 18 Vials stored at 30° C.: high molecular weight species, percent.Composition Day 36 Day 50 Day 64 P 0.33 0.41 0.46 Q 0.29 0.39 0.43 R0.29 0.38 0.43

TABLE 19 3-mL cartridges stored 14 days at 37° C. with shaking. % OtherRelated Composition % HMW Units/ml Substances P 0.33 98.24 1.32 Q 0.3497.88 1.54 R 0.30 97.66 1.59

An additional stability study is performed wherein formulationscontaining insulin lispro and different concentrations of citrate,treprostinil, zinc and other excipients are subjected to differentconditions. Twenty-four formulations of citrate-containing formulationsand two control formulations which do not contain citrate are prepared.The concentrations of citrate, magnesium chloride, Zn, total chloride,treprostinil and poloxamer in the formulations are indicated in Table 20below.

TABLE 20 Formulations Tested Total Citrate MgCl₂ Total Zn ChlorideTreprostinil Poloxamer No. (mM) (mM) (mM) (mM) (μg/mL) 188% S 0 0 0.3 100 0 T 0 0 0.3 20 0 0 U 12 2.5 0.4 10 2 0 V 12 2.5 0.8 13 0 0 X 12 7.50.4 20 0 0 Y 12 7.5 0.8 23 2 0 Z 15 5 0.6 20 1 0 AA 18 2.5 0.4 10 0 0 BB18 2.5 0.8 13 2 0 CC 18 7.5 0.4 20 2 0 DD 18 7.5 0.8 23 0 0 EE 15 5 0.616 1 0.045 FF 15 5 0.6 16 1 0.045 GG 15 5 0.6 25 1 0.045 HH 15 5 0.6 301 0.045 II 15 5 0.6 50 1 0.045 JJ 15 5 0.6 100 1 0.045 KK 15 5 0.6 20 10.06 LL 12 2.5 0.4 10 0 0.09 MM 12 2.5 0.8 13 2 0.09 NN 12 7.5 0.4 20 20.09 OO 12 7.5 0.8 23 0 0.090 PP 18 2.5 0.4 10 2 0.09 QQ 18 2.5 0.8 13 00.09 RR 18 7.5 0.4 20 0 0.090 SS 18 7.5 0.8 23 2 0.090

In order to prepare the formulations, a lispro concentrate stocksolution is prepared containing 200 units insulin lispro/mL, 24.2 mgglycerin/mL, 6.30 mg metacresol/mL, and 0.6 mM zinc in water for bulksterile operations. This stock solution is pH adjusted with hydrochloricacid to dissolve the insulin lispro and then adjusted with sodiumhydroxide to pH of 7.40.

The citrate-containing formulations are prepared by diluting the lisproconcentrate with the appropriate volumes of excipient stock solutions:sodium citrate, magnesium chloride, zinc oxide, treprostinil, andpoloxamer 188. To achieve target chloride content, a stock solution ofsodium chloride is added to some formulations. The solution is pHadjusted to 7.30-7.50 with hydrochloric acid or sodium hydroxidefollowed by q.s. with water for bulk sterile operations.

The non-citrate formulations are prepared by diluting the lisproconcentrate with the appropriate volumes of water for bulk sterileoperations or water for bulk sterile operations and sodium chloridesolution. The solution is pH adjusted to 7.30-7.50 with hydrochloricacid or sodium hydroxide followed by q.s. with water for bulk sterileoperations.

The formulations are sterile filtered and then volumetricallytransferred to 10 mL glass vials with a 10.3 mL fill, stoppered, andcrimp sealed. Twenty vials are filled per batch.

The study includes subjecting vials of each formulation to one of threedifferent conditions, as described below.

TABLE 21 Condition Description Control (n = 5) 5° C. static uprightstorage for 39 days Thermal stress (n = 5) Abbreviated shipping stresssimulation followed by 30° C. static upright storage for 32 daysCumulative Stress (n = 5) Abbreviated shipping stress simulationfollowed by 32 day simulated patient in-use with 30° C. upright storagebetween dosing

As indicated in the table above, the thermal stress and cumulativestress conditions include first subjecting the vials to an abbreviatedshipping stress simulation, which is performed as described inInternational Safe Transit Association (ISTA) Procedure 3A (2008). Forthe thermal stress condition, the vials are then placed into staticupright storage at 30° C. For the cumulative stress condition, the vialsare then subjected to a patient in-use simulation. The patient in-usesimulation is performed by withdrawing 8 units of air into any insulinsyringe, inserting the needle into the vial while the vial is in theupright position, ensuring that the needle tip does not touch theinsulin solution, injecting air into the vial, inverting the vial andsyringe, withdrawing 8 units of the product solution, eliminating anyair bubbles in the syringe by slowly moving the plunger to push thebubble back into the vial (repeating if necessary) and adjusting thesyringe plunger so that the final dosage is 8 units, removing thesyringe, and placing the vial back into the 30° C. incubator. Thisprocess is repeated for three doses per day for 32 days. When not beingdosed, the vials are stored upright at 30° C.

Chemical stability is tested by size exclusion chromatography (SEC) andreverse phase HPLC (RP-HPLC). RP-HPLC analysis is performed to assessprotein purity in each formulation using a UV detector at 214 nm. Eachsample (5 μL) is separated using a Halo Peptide ES-C18, 4.6×150 mm2.7-Micron column (Part #92124-702) at 40° C. with a flow rate of 1.0mL/minute and mobile phase A (50 mM sulfate, pH 2.3+20% acetonitrile(v/v)) and mobile phase B (50 mM sulfate, pH 2.3+50% acetonitrile(v/v)). Gradient of mobile phase B at 0, 2, 17, 22, 25, 29, 29.5, and 35minutes is 25, 25, 27, 40, 90, 90, 25 and 25%, respectively. Insulincontent is calculated by combining the integrations of the insulin mainpeak and B3 peak areas then dividing by an insulin lispro standard. ForSEC, each sample (50 μL) is separated using a Sepax Zenix-C SEC-80,7.8×300 mm, 3 μm particles column (catalog #233080-7830) at 5° C. and aflow rate of 1.0 mL/minute with isocratic elution of mobile phase (0.1%TFA, 50% ACN) over a run time of 25 minutes. Percentage of high moleculeweight polymer (% HMWP) is calculated by integrating the total percentarea of all peaks eluting prior to the main peak. One vial each fromformulations PP and QQ subjected to the cumulative stress conditionscould not be sampled due to particulate formation. All other vials weretested, and all results were within insulin lispro acceptance criteriafor both potency (95-105 U/mL) and HMWP (NMT 1.5%).

Physical stability is assessed by visual appearance testing in whichsamples are visually inspected. Samples are graded as pass that areclear, colorless, and essentially free of visible particles. All samplesof all formulations pass visual appearance testing at all time points inthe control and thermal stress conditions. Visual appearance testingresults for the cumulative stress condition are provided in table 22below.

TABLE 22 24 Days 28 Days 32 Days Formulation 21 Days (n = 5) (n = 5) (n= 5) (n = 5) S 100% Pass 100% Pass 100% Pass 100% Pass T 100% Pass 100%Pass  80% Pass 100% Pass U 100% Pass 100% Pass 100% Pass 100% Pass V100% Pass 100% Pass 100% Pass 100% Pass X 100% Pass 100% Pass 100% Pass100% Pass Y 100% Pass 100% Pass 100% Pass 100% Pass Z 100% Pass 100%Pass 100% Pass 100% Pass AA 100% Pass 100% Pass 100% Pass 100% Pass BB100% Pass 100% Pass 100% Pass 100% Pass CC 100% Pass 100% Pass 100% Pass100% Pass DD 100% Pass 100% Pass 100% Pass 100% Pass EE 100% Pass 100%Pass  40% Pass  0% Pass FF 100% Pass 100% Pass 100% Pass  0% Pass GG100% Pass  80% Pass  0% Pass  0% Pass HH 100% Pass 100% Pass  0% Pass 0% Pass II 100% Pass 100% Pass  0% Pass  0% Pass JJ 100% Pass 100% Pass 0% Pass  0% Pass KK 100% Pass 100% Pass  0% Pass  0% Pass LL  60% Pass 60% Pass  40% Pass  0% Pass MM 100% Pass 100% Pass 100% Pass  20% PassNN 100% Pass 100% Pass 100% Pass  80% Pass OO 100% Pass 100% Pass 100%Pass 100% Pass PP  20% Pass  0% Pass  0% Pass  0% Pass QQ  80% Pass  80%Pass  20% Pass  0% Pass RR 100% Pass 100% Pass 100% Pass  0% Pass SS100% Pass 100% Pass 100% Pass  0% Pass

All vials of formulations U, V, X, Y, Z, AA, BB, CC DD and OO pass thecumulative stress testing through day 32 of the patient in-usesimulation. All vials of formulations FF, MM, NN, RR and SS pass thecumulative stress testing out through day 28. All vials of formulationsEE, HH, II, JJ and KK pass through day 24. All vials of formulation GGpass out through day 21.

The studies described above support that the compositions are chemicallyand physically stable under refrigerated and thermal stress conditions,and that certain compositions of the present invention are sufficientlystable for commercial use even under simulated in use testing out to 32days.

Sequences

Human insulin A-chain (SEQ ID NO: 1)Gly Ile Val Glu Gln Cys Cys Thr Ser Ile CysSer Leu Tyr Gln Leu Glu Asn Tyr Cys Asn. Human insulin B-chain(SEQ ID NO: 2) Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu ArgGly Phe Phe Tyr Thr Pro Lys Thr.  Insulin lispro B-chain (SEQ ID NO: 3)Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu ArgGly Phe Phe Tyr Thr Lys Pro Thr.  Insulin aspart B-chain (SEQ ID NO: 4)Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu ArgGly Phe Phe Tyr Thr Asp Lys Thr. Insulin glulisine B-chain(SEQ ID NO: 5) Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu ArgGly Phe Phe Tyr Thr Pro Glu Thr.

We claim:
 1. A pharmaceutical composition comprising: a. an insulin; b.citrate, in a concentration from about 5 to about 25 mM; c.treprostinil, in a concentration from about 0.04 to about 20 μg/mL; d. apreservative; and e. zinc, in a concentration sufficient to provide atleast 2 zinc ions per six molecules of insulin; and having a pH of about7.0 to about 7.8 at room temperature, wherein the pharmaceuticalcomposition provides for an uptake of insulin into the blood, asmeasured by early ½ Tmax, that is at least 20% more rapid than forcompositions which contain the same insulin but which do not containcitrate or treprostinil; and wherein the composition does not compriseEDTA.
 2. The pharmaceutical composition of claim 1 wherein the zincconcentration is from about 0.2 to about 2 mM.
 3. The pharmaceuticalcomposition of claim 1, wherein the zinc concentration is from about 0.6to about 0.8 mM.
 4. The pharmaceutical composition of claim 3, furthercomprising magnesium chloride.
 5. The pharmaceutical composition ofclaim 4 wherein the concentration of magnesium chloride is about 5 mM.6. The pharmaceutical composition of claim 5 wherein the insulin isinsulin lispro.
 7. The pharmaceutical composition of claim 6 wherein theinsulin lispro is present in a concentration from about 100 to about 200U/mL.
 8. The pharmaceutical composition of claim 7 further comprisingglycerol.
 9. The pharmaceutical composition of claim 8 wherein theconcentration of glycerol is about 12 mg/mL.
 10. The pharmaceuticalcomposition of claim 9 wherein the preservative is m-cresol.
 11. Thepharmaceutical composition of claim 10 wherein the concentration ofm-cresol is about 3.15 mg/mL.
 12. The pharmaceutical composition ofclaim 11 wherein the pharmaceutical composition is stable for up to 28days at temperatures of up to 30° C.
 13. The pharmaceutical compositionof claim 1 wherein the pharmaceutical composition does not contain anyadditional vasodilatory agent.
 14. The pharmaceutical composition ofclaim 1 wherein the pharmaceutical composition does not contain anyoligosaccharides.
 15. The pharmaceutical composition of claim 1 wherein:a. the insulin is insulin lispro, and is present in a concentration fromabout 100 to about 200 U/mL; b. citrate is in a concentration of about15 mM; c. treprostinil is in a concentration of about 1 μg/mL; d. thepreservative is m-cresol, and is present in a concentration of about3.15 mg/mL; and e. zinc is in a concentration from about 0.6 to about0.8 mM; and wherein the pharmaceutical composition further comprises: f.glycerol, in a concentration of about 12 mM; and g. magnesium chloride,in a concentration of about 5 mM; and wherein the pharmaceuticalcomposition does not contain EDTA, any additional vasodilatory agents orany oligosaccharides; and wherein the pharmaceutical compositionprovides for an uptake of insulin into the blood, as measured by early ½Tmax, that is at least 20% more rapid than for compositions whichcontain the same insulin but which do not contain citrate ortreprostinil; and wherein the pharmaceutical composition is stable for(i) at least 24 months at 2-8° C., and (ii) up to 28 days attemperatures of up to 30° C.
 16. A method of treating diabetescomprising administering to a human in need thereof an effective dose ofthe pharmaceutical composition of claim
 1. 17. An article of manufacturecomprising the pharmaceutical composition of claim
 1. 18. The article ofmanufacture of claim 17 which is a multi-use vial.
 19. The article ofmanufacture of claim 17 which is a multi-use cartridge.
 20. The articleof manufacture of claim 17 which is a re-usable pen injector.
 21. Thearticle of manufacture of claim 17 which is a disposable pen device. 22.The article of manufacture of claim 17 which is a pump device forcontinuous subcutaneous insulin infusion therapy.